Light activation of neurons is a growing field with applications ranging from basic investigation of neuronal systems to the development of new therapeutic methods. Several light activation approaches have been demonstrated using light sensitive membrane proteins [1, 2]. A different technique consists of using quantum dots (QDs) as artificial chromophores that can be placed at close proximity to cells and mediate electrical activation. Only few studies have indicated the potential of QDs in the realm of neuron photo-activation [3, 4]. It was demonstrated that HgTe particles could be used for light-induced activation of neurons in culture. The nanoparticles were deposited using a layer-by-layer (LBL) method on an ITO substrate and neuron culture was grown onto this surface. 532 nm laser illumination at 800 mW/cm2 intensity was used (more than an order of magnitude higher than daylight intensity (˜30 mW/cm2)), resulting in successful neuronal stimulation. Invitrogen Corp. [5] has exploited the photoelectronic properties of QDs and developed a nanostructured biocompatible interface composed of stacks of semiconductor nanoparticles coated with an adhesion protein layer. The feasibility to interface with neurons through light activation has been demonstrated by [6, 7, 8].
General Description
Improving charge transfer efficiencies of photo-excited chromophores is at the focus of research conducted in the field of solar cell technology. Such solar cells include QD-coated electrodes embedded in electrolyte containing charge donors. In the presence of light, charges are injected from a QD matrix into the underlying support electrode and a direct current between a counter electrode in the solution and the QD-coated electrode is obtained. The manner by which the QDs are coupled to the electrode and to the solution are of utmost importance as the environment can affect resonance quenching or alternatively enhance charge transport processes. It is also likely that the effective band structure of the QDs is affected, giving rise to improved or suppressed photocurrent.
One particular approach to control these processes and to enhance the photocurrent is to use single wall carbon nanotubes as a linker between the QDs and the electrode. Organic coupling agents can be used to link the QDs to CNTs, requiring harsh chemical treatment of the CNTs to allow for their chemical functionalization. Another strategy for coupling QDs to CNTs uses a one-pot synthesis, in which CdSe QDs are grown in the presence of the CNTs leading to composites in which multiple QDs decorate the CNT along its axis. In this case, unlike the organic linking scenario, the QDs are closely adjacent to the CNTs, and this is expected to provide strong coupling between the two systems.
The present invention provides a novel photoelectrical device for efficient transmission of electrical signals to a neuron, so that the threshold for generating action potentials (impulses) is reached. This photoelectrical device comprises one or more charging units configured for coupling to and stimulating one or more neurons by charge accumulation, the charging unit comprising: a nanostructure based electrode having a surface, which has a predetermined developed surface area for coupling to a neuron and which carries a plurality of spaced-apart photosensitive regions (e.g. QDs) interfacing with a biocompatible macromolecule for tuning the relative energy levels between the photosensitive regions and the electrode, as well as for directing the spatial polarity of charge separation; the surface of the nanostructure based-electrode being thereby electrically chargeable and dischargeable in response to light excitation of the photosensitive regions, the charges stimulating the neuron when coupled to the surface, and thus enabling the transmission of electrical signals to the neuron.
In particular, the device of the present invention may be used for neuro-chip technology and more specifically for retinal implant technology.
Multielectrode arrays (MEAs) may be used to interface with neural assemblies, both for stimulation and recording purposes. In particular MEAs may be used for neural prosthetic devices to artificially restore impaired neural function (e.g. for vision, hearing and limb movement). One key issue is the biocompatibility of the electrode-tissue interface which must not only allow good electrical recording and stimulation performance, but also encourage strong cell adhesion and proliferation so that the electrodes remain effectively long-term coupled with the neurons. For optimal electrical coupling, the electrode surface must be as rough as possible so that it effectively enlarges the surface area. Independently, to achieve good biological coupling, the surface must be cell-adhesive. Interestingly, these two requirements can be attained concurrently if extremely rough and conducting surfaces are used [12]. Such surfaces can function both as excellent electro-chemical electrodes and substrates for neuronal growth [13].
The device is therefore activated (e.g. pumped) optically and the device output is in the form of an electric charge. It should be understood that although the device utilizes a photoelectric effect, the device is configured to use charge accumulation and not photocurrent generation as the device output.
In some embodiments, the nanostructure based-electrode includes carbon nanotubes (CNTs) having a predetermined developed surface area (i.e. specific outer surface) for coupling to a neuron (e.g. to obtain a strong neuron-carbon nanotubes affinity). It should be understood that the term “nanostructure electrode” has to be interpreted in a broadest manner in which the thickness of the electrode is in the order of micrometers and the diameter of the electrode is in the order of few micro-meters. The specific outer surface of the CNT enhancing the coupling of CNTs to neurons is characterized by a relatively long length of the nanotube and by the roughness of the outer surface. For example, the roughness of the electrode can be characterized by an electrode effective area 1,000 larger than the geometrical area of the electrode. Examples of such coupling and techniques for measuring the degree of coupling are described in Raya Sorkin, Alon Greenbaum, Moshe David-Pur, Sarit Anava, Amir Ayali, Eshel Ben-Jacob, and Yael Hanein, Process entanglement as a neuronal anchorage mechanism to rough surfaces. Nanotechnology, 20 (2009) 015101, which is incorporated herein by reference with respect to this specific example. Accordingly, the length of each nanotube is selected to be in the range of about 1 and 50 μm.
The CNTs operate as mediator to enhance the efficiency of the coupling between QDs and neurons. CNTs provide a very effective adhesive material for neuronal proliferation and facilitate both the strong coupling between the cells and the surface and the direct electrical recordings. The use of CNTs also contributes to the mechanical and electrical interfacing because they are chemically inert and robust against mechanical damage and easy to produce. Moreover, it should be noted that CNTs improve the performance of MEAs due to their many beneficial properties such as mechanical stability, chemical durability, good electrical conductance and their bio-compatibility.
It should be understood that CNTs possess unique electronic and mechanical properties. The CNT-based electronic device is dictated by its size and chirality to be either semiconducting or metallic, providing flexible tuning of the electrical properties. Moreover, CNTs are also highly neuronal-adhesive material and can serve as a good substrate for neural growth. Electrical stimulation of neurons grown on the CNTs in multi-electrode array (MEA) architecture is highly efficient.
QDs are versatile absorbing and emitting chromophores whose optical and electronic properties can be tuned by modifying the size, shape and composition of the particles. In particular, quantum confinement effects provide particles with band gaps covering the UV-VIS-NIR range. Moreover, QDs are photo and electro-active and can generate photocurrents. Diverse chemistries can be applied to the QDs surfaces to tailor them to be biocompatibility [14]. Some examples of QDs include nanoparticles selected from the periodic table group II-VI semiconductors and III-V semiconductors. Group II-VI semiconductors includes at least one of CdSe, CdTe, CdS, ZnSe, Group III-V semiconductors includes at least one of InP, GaAs. InP nanoparticles, in particular, offer very good spectral coverage in the visible range, and are therefore suitable for stimulation of neurons imitating retinal function.
In some embodiments, the quantum dot is selected from core/shell nanoparticles, heterostructured nanoparticles and a combination thereof. The core/shell nanoparticles include CdSe/CdS core/shell, CdSe/CdTe core/shell, CdSe/ZnS core/shell. The heterostructured nanoparticles include CdSe/ZnSe, InP/CdS, InP/ZnSe and other combinations. The heterostructured nanoparticles may have an elongated portion or being in the form of a nanorod.
Therefore, the present invention overcomes the low efficiency of conventional cell activation with light methods, by providing new efficient hybrid devices interfacing between chromophores and CNT to achieve efficient chromophore-neuron coupling including the cell-surface coupling as well as for electro-chemical interfacing efficiency.
It should be understood that to provide an efficient coupling between the carbon nanotubes and the QDs, the selection of appropriate chemical and electrical properties of the biocompatible macromolecules is critical. To this end, the biocompatible macromolecules and the QDs have to be linked by electrostatic or covalent interaction.
The biocompatible macromolecules are selected amongst polymers, biopolymers, macromolecules and other large molecules which, on one hand, are non-toxic, chemically inert, and substantially non-immunogenic to a living tissue, in vivo, ex vivo, in a human tissue or a tissue of a non-human, and, on the other, are conductive and capable of association with both the QDs and the CNTs.
The biocompatible macromolecules are used to tune the relative energy levels of the chromophores and the CNT as well as a means to direct the spatial polarity of charge separation.
The biocompatible macromolecule may be selected from polymers and biopolymers such as melanin, polyaniline (PANI), polypeptide, polypyrrole, polypeptide (linear, branched and looped conformations), poly-l-lysine, cellulose acetate, ethylene vinyl alcohol copolymer or any pre-polymer (e.g., a subset of lower molecular weight polymers or monomers, oligomers thereof), blend, mixture and hybrid thereof.
The macromolecule may be in the form of a polymer film (e.g. initially selected conductive polymer (CP) film or initially non-conductive converted into conductive during the device manufacture). The film may be located between the CNT and the QD (i.e. CP encapsulates the QD on top of the CNT) and may be associated therewith any one physical or chemical bond such as covalent, electrostatic, π-bonding, hydrogen-bonding interactions and van der Waals. The association of the biocompatible macromolecule should be selected or adapted to be either covalent or electrostatic interaction (association) with the QD and either π-bonding interaction or van der Waals interaction with the CNT. The interaction may involve bond formation between surface groups of the QD and the CNT having surface groups, which in some embodiments, include use of known bio-conjugation schemes such as avidin-biotin interaction. The macromolecule may alternatively be in the form of a coating on at least a portion of an outer surface of the QD (directly coupled to the CNT) and/or on any space region between the QDs on the CNT's surface. The thickness of such films is in the order of nanometers to ten nanometers and even to several microns.
As mentioned above, the polymer selected to be used in the device may not be inherently conductive, but may become conductive once the device is formed.
Generally, the device of the invention may be constructed directly from any one biocompatible macromolecule, e.g., biocompatible polymer, or indirectly through polymerizing, either in situ or prior to application, of pre-polymers, such as monomers or oligomers, which may or may not be in association (e.g., already bonded) with one or more of the QDs and/or the CNTs. Polymerization of the pre-polymer may be carried out employing any one method of polymerization. Non-limiting examples are electropolymerization, chemical polymerization photochemical polymerization and enzymatic polymerization, plasma polymerization, as known in the art. The polymerization may be initiated by anyone catalyst, as known in the art, or by actinic radiation. For example, where the biocompatible polymer is polyaniline, the monomer to be polymerized, e.g., via electropolymerization is aniline. In such a case, a charge-transport layer is used to couple the QDs and CNTs. The CNTs are wrapped with nanolayers of conducting polymers (CP) leading to a significant drop in the electrode impedance while preserving neuro-compatibility.
In other embodiments, one or more of the photosensitive regions are encapsulated within a layer of a conductive polymer, such that the electrical contact between the photosensitive region and the nanostructure based-electrode is through the polymer.
The invention also provides a process for the manufacture of a photoelectrical device for stimulating a neuron. The method comprises selecting one or more carbon nanotubes to have a predetermined developed surface area for coupling to a neuron; the one or more carbon nanotubes being configured and operable as electrodes; associating a plurality of photosensitive quantum dots with each carbon nanotube; the obtained nanotubes associated with the photosensitive quantum dots being electrically chargeable and dischargeable in response to light excitation of the quantum dots, the charges stimulating the neuron when coupled to the surface; and coating with a polymer (as defined and characterized hereinabove) at least a portion of the carbon nanotube associated with the plurality of photosensitive quantum dots for tuning the relative energy levels between the photosensitive quantum dots and the carbon nanotube, as well as for directing the spatial polarity of charge separation.
The distribution of the QDs over the surface of the CNT may also depend, among other parameters, on the reaction conditions, as well as on the nature and chemical composition of the QDs, and on their size. The plurality of QDs may be arranged in a spaced-apart arrangement over at least one portion of the nanotube surface, where each QD or groups of two or more QDs (e.g. aggregates) are positioned at a certain distance from another QD. Alternatively, the QDs may be arranged in an unordered fashion over the surface of the CNT.
The association of the plurality of QDs with the CNTs, in any arrangement, may be achieved by depositing the QDs onto the carbon nanotubes, e.g., by electro-deposition. Alternatively, the QDs may be synthesized in the presence of the CNTs, thereby achieving direct association.
The invention further provides a process for manufacturing a photoelectrical device for stimulating neurons. The method comprises providing one or more photosensitive quantum dots coated with at least one pre-polymer (e.g., a monomer of a polymer); and polymerizing the pre-polymer (e.g., in situ) in the presence of a carbon nanotube selected to have a predetermined developed surface area for coupling to a neuron; the one or more carbon nanotubes being configured and operable as electrodes, the obtained nanotubes associated with the photosensitive quantum dots coated with polymer being electrically chargeable and dischargeable in response to light excitation of the quantum dots, the charges stimulating the neuron when coupled to the surface, thereby providing a photoelectrical device for stimulating neurons. In some embodiments, the pre-polymer is electropolymerizable. In this case, the step of polymerizing the pre-polymer comprises electropolymerization.
The invention is further concerned with a process of manufacturing of a photoelectrical device operable in biological conditions for stimulating neurons. The process comprises providing a substrate carrying at least one carbon nanotube, contacting the substrate with an aqueous or organic solution (or a mixture of organic solvents such as alcohols) comprising a plurality of charged (negatively or positively) quantum dots and with a solution (e.g. organic solution) containing charged biocompatible macromolecules (positively or negatively charged) or mixtures thereof; thereby to permit association between the nanotube, the quantum dots and a coating layer of the biocompatible macromolecules.
Therefore, there is provided a method of making a photoelectrical device for stimulating neurons. The method comprising: providing a substrate carrying one or more carbon nanotubes selected to have a predetermined developed surface area for coupling to a neuron; the one or more carbon nanotubes being configured and operable as electrodes; providing two solutions: a quantum dot solution containing charged photosensitive quantum dots; and a solution containing charged biocompatible macromolecules; the biocompatible macromolecules being configured for tuning the relative energy levels between the photosensitive quantum dots and the carbon nanotube, as well as for directing the spatial polarity of charge separation; and dipping the substrate first in one of the two solutions and then with the other solution to yield a nanotube carrying quantum dots and coated with biocompatible macromolecules, thereby providing a photoelectrical device for stimulating neurons.
In some embodiments, the substrate is first contacted with the aqueous solution comprising a plurality of charged quantum dots and thereafter with the solution containing charged biocompatible macromolecules.
In some embodiments, the substrate is first contacted with the aqueous solution comprising a plurality of charged quantum dots and thereafter is plasma polymerized with biocompatible macromolecules.
In some embodiments, the substrate is first plasma polymerized with biocompatible macromolecules, then contacted with the aqueous solution comprising a plurality of charged quantum dots and thereafter is plasma polymerized again with biocompatible macromolecules.
In other embodiments, the substrate is first contacted with the solution containing charged biocompatible macromolecules and thereafter with the aqueous solution comprising a plurality of charged quantum dots. The step of contacting between the substrate and the solutions may be performed by way of dipping, which may be repeated any number of times.
In some embodiments, the biocompatible macromolecules and the quantum dots are oppositely charged.
The present invention further provides a method of stimulating a neuron. The method comprises providing a photoelectrical device according to the present invention; placing the photoelectrical device in the vicinity of a neuron to be stimulated; and irradiating the device with light to thereby enable neuron stimulation.
In some embodiments, the method involves growing the neuron in the vicinity of the photoelectrical device.
In some embodiments, irradiating the photoelectrical device with light comprises selecting power irradiations and wavelength of light to control a stimulation level of the neuron.
The present invention further provides a method for retinal stimulation to create artificial vision keeping the output cells of the retina intact. The method comprises providing a photoelectrical device according to the present invention and placing the device in the vicinity of a neuron to be stimulated. Once signal indicative of input light (image) enters the retina through the device implanted in the retina, information is transmitted from the retina to the visual centers of the brain. Thus, the invention provides an artificial retinal device suitable to electrically stimulate the retina in the eye to produce artificial vision.
In this connection, it should be understood that degenerative retinal dystrophies are a major cause for blindness among the aging population. The common phenotype of these diseases is photoreceptor degeneration, whilst retinal ganglion cells (RGCs) (the output cells of the retina), remain intact and can still transmit information to the visual centers of the brain. Consequently, retinal implantable prosthetic devices can be used to stimulate RGCs directly, circumventing the degenerated photoreceptor pathway.